A major objective of this funding cycle was to explore the feasibility of using electrochemical microsensors (polarographic probes- Serp) in a self-referencing modality. We began this development in the previous cycle but this year has seen accumulating evidence for the power of this new technique. Our already existing ion-selective self-referencing (Seris) systems are limited by the range of suitable commercially available ionophores. These liquid membranes must exhibit both adequate selectivity and rapid enough response times. These constraints have limited us to calcium, chloride, hydrogen and sodium detection. To expand our repertoire of molecular detection techniques we targeted the characteristic reduction oxidation potentials of certain molecules as an alternative detection method which should benefit from the same drift reduction as the old voltage and Seris systems. We began our development by targeting oxygen a clearly important metabolic signal that prior to our work could not be measured from small single cells let alone with square micron spatial accuracy. We approached the problem of oxygen detection using the translational movement of a Whalen type microelectrodes (tip diameter = 2-3?m) through a gradient at a known frequency and between known points. Accounting for the linear calibration of the electrode, the differential electrode output is converted into a directional measurement of flux using the Fick equation. Operational characteristics of the technique were determined using artificial gradients produced outside of artificial sources of oxygen flux created using a micropipette and gases of known oxygen concentrations. The experiments showed that oxygen flux values obtained from the self-referencing oxygen microelectrode matched derived values calculated from static measurements of oxygen obtained outside of the artificial source. This reporting year has seen the further refinement of our source modeling as well as the construction of our own electrodes. In addition we have begun to routinely apply this technique to small single mammalian cells held at 37oC. Thistechnique has now been applied to various experimental systems including the study of neuronal metabolism (Smith, Dubinsky), metabolism in oocytes and embryos (Keefe, Porterfield, Sardet), pollen tubes (Robinson) and the metabolic regulation of insulin secretion in ?-cells (Corkey).